A defining feature of Type 2 Diabetes Mellitus (T2DM) is chronic hyperglycemia, which, is driven by excessive hepatic gluconeogenesis. Mitochondrial pyruvate is required for approximately 90% of gluconeogenesis. Pyruvate entry into the mitochondrial matrix requires transit through the Mitochondrial Pyruvate Carrier (MPC). Because the MPC gates mitochondrial pyruvate uptake, the MPC may be a critical regulatory point for gluconeogenic carbon flux and a potential T2DM therapeutic target. The goal of this research proposal is to elucidate the mechanisms regulating MPC activity in the liver and to determine how this activity is altered in T2DM. We hypothesize that the liver MPC activity is pathologically elevated in T2DM, thereby enabling or driving excessive gluconeogenesis. Furthermore, reduction of MPC activity during T2DM may treat hyperglycemia. Therefore, for Specific Aim 1 we will seek to: 1) Determine if liver MPC activity is misregulated during T2DM; and 2) Determine if acute genetic reduction in liver MPC activity ameliorates hyperglycemia observed in T2DM. The goals of this research are two-fold. First, we will determine whether under conditions of T2DM the MPC activity is misregulated in response to refeeding after fasting; and second, if acute homo- and heterozygous liver-specific MPC1 knockout reduces the excessive gluconeogenesis and hyperglycemia observed during T2DM, thereby testing whether the MPC has promise as a therapeutic target for T2DM. Virtually all metabolic enzymes are regulated; many by multiple mechanisms. The MPC comprises two paralogous subunits, MPC1 and MPC2, which may be differentially regulated to control overall MPC function. Given the pivotal node the MPC occupies undergirding mitochondrial and cellular metabolism, we hypothesize that it is also regulated. Evidence supporting this hypothesis includes early studies utilizing crude mitochondrial extracts suggesting that MPC activity was increased by glucagon and decreased by insulin. However, when these studies were performed, a targeted molecular characterization was impossible because the identity of the MPC was unknown. The recent discovery of the molecular identity of MPC provides the first opportunity to investigate the molecular mechanisms regulating MPC function. For Specific Aim 2 we will utilize hepatocyte cell culture models to determine whether insulin and glucagon regulate the MPC activity in liver cells: 1) By transcriptional and or protein turn-over mechanisms that regulate MPC abundance and total activity; and 2) By phosphorylation or acetylation of MPC1 and MPC2 protein to regulate MPC specific activity. We expect to observe that MPC1 and MPC2 are regulated by multiple mechanisms which may be altered in response to T2DM. Determining the potentially unique mechanisms regulating MPC1 and MPC2 may be critically important for understanding the overall function of the MPC complex.